Try to talk to your colleague about his/her application. If your colleague do not require a native protein, you can just use your current column purified products and do SDS-PAGE, followed by excise the band and do electro elution. After the electro elution, you can concentrate the protein by using vivaspin protein concentrator (column with membrane). Do a protein quantification (BCA, Bradford etc...), and dilute to the concentration you want.

LOLx, I'm also a dummies as well...

-adrian kohsf-

rpjkmust916 on Thu Feb 17 02:22:57 2011 said:

Thanks proteaMatt and mdfenko,

Now my knowledge in protein is increasing, may I ask more

mdfenko on Tue Feb 15 21:11:57 2011 said:

the two bands you see could be subunits of the protein. are they in the same ratio for each sample? do you know the native molecular weight of the protein? how do these bands compare? do they both stain in a western blot? if so, are you using polyclonal or monoclonal antibodies?

the lower band could be a fragment of your protein of interest.

May I know, what do you mean 'in the same ratio for each sample'?
I have not learn to do the western blot yet
Is it possible to identify it as a fragment if I run western blot?

About the native molecular weight of the protein, let me give the details
It is a nuclear receptor protein with 280-486 aa, cloned into PET 32a Rosetta.
My senior told me the molecular weight of this protein is 43KDa.
The calculation she showed me was like this,
- 206 aa x 110 Da = 22660 so ~23KDa
- 23KDa + 20 = 43KDa

Actually, I don't understand But I'm afraid to ask for more because we do have language barrier and she seems like not in good condition lately.

mdfenko on Tue Feb 15 21:11:57 2011 said:

you should always have an idea of how much protein you load in each lane but that is not really necessary to determine percent purity.

you can evaluate the lanes by either scanning them or by imaging them (take a picture) and determine the relative amount of each band in each lane (you can use a program like imagej to evaluate the image).

Thanks for the explanation.
There is no scanning machine for the gel in the lab. I only scanned the gel with scanner that attached with the computer.

Thank you again and again.....

the scanned image of the gel can be brought into a program like imagej and the lanes can be evaluated like they were scanned. each band can be integrated and given a value relative to the amount of stain incorporated (you can, if you know how much protein was loaded, determine the actual mass for each band). you can then determine the percent purity based on the whole lane and the ratio of the two bands of interest.

the ratio would be important to determine if the two bands are subunits of the same protein, if they maintain the same ratio for all samples then they may be. from what you say in the post about the formula weight of your protein, they are probably not subunits.

you can determine if the lower band is a fragment of the upper band by western blot if the remaining protein contains the epitope(s) recognized by the antibody.

-mdfenko-

mdfenko on Thu Feb 17 21:39:24 2011 said:

the scanned image of the gel can be brought into a program like imagej and the lanes can be evaluated like they were scanned. each band can be integrated and given a value relative to the amount of stain incorporated (you can, if you know how much protein was loaded, determine the actual mass for each band). you can then determine the percent purity based on the whole lane and the ratio of the two bands of interest.

the ratio would be important to determine if the two bands are subunits of the same protein, if they maintain the same ratio for all samples then they may be. from what you say in the post about the formula weight of your protein, they are probably not subunits.

you can determine if the lower band is a fragment of the upper band by western blot if the remaining protein contains the epitope(s) recognized by the antibody.

Thanks mdfenko,
I will learn how to use the ImageJ

Adrian,

Thank you for ur advice....

-rpjkmust916-

Welcome, hope this helps.

-adrian kohsf-

some might be satisfied with CCB-stained gels to state purity however staining with silver or nanogramm-sensitive dyes is the correct method to state purity on the gel level...

rpjkmust916 on Fri Feb 11 08:46:25 2011 said:

Hi Everyone,

This is my second post in this section.
Attached herewith is the pic of my SDS-Page of protein purification that I've done.
The 8,9 and 10 column were the protein that I need to purify.
Based from the bands that appear, I would like to confirm whether it is pure or not. Any advice,suggestion and ideas are welcome.
Thank you.

-Inmost sun-

Inmost sun on Sat Feb 19 15:03:14 2011 said:

some might be satisfied with CCB-stained gels to state purity however staining with silver or nanogramm-sensitive dyes is the correct method to state purity on the gel level...

May I ask about the electroelution?
I've read several papers about electroelution...looks like it require some specific apparatus. Is there any simple method that I can use to run electroelution?
Any suggestions, please?

-rpjkmust916-

rpjkmust916 on Tue Feb 22 12:24:07 2011 said:

Inmost sun on Sat Feb 19 15:03:14 2011 said:

some might be satisfied with CCB-stained gels to state purity however staining with silver or nanogramm-sensitive dyes is the correct method to state purity on the gel level...

while silver stain is very sensitive and qualitative, it is too variable between different proteins to use for quantitation (and, therefore, percent purity). coomassie brilliant blue r-250 (not as variable as g-250) is well suited for this purpose. you may need to load more to see minor contaminants but they are minor and will not significantly influence percent purity determinations

May I ask about the electroelution?
I've read several papers about electroelution...looks like it require some specific apparatus. Is there any simple method that I can use to run electroelution?
Any suggestions, please?

all you need is dialysis tubing, horizontal gel apparatus and a suitable buffer

-mdfenko-

Thanks mdfenko,

Can I use the running buffer that I've used to purify the protein with IMAC column to do the electroelution?
Or do I have to find a different type of buffer?

May I know, how much the concentration of target protein that you've usually recover from electroelution?

Thank you very much for spending time in answering my questions.

-rpjkmust916-

rpjkmust916 on Wed Feb 23 01:34:02 2011 said:

Can I use the running buffer that I've used to purify the protein with IMAC column to do the electroelution?
Or do I have to find a different type of buffer?

standard electrophoresis buffers work well, others you may have to try yourself

May I know, how much the concentration of target protein that you've usually recover from electroelution?

concentration depends on how much protein is in the band being eluted and the volume of buffer in the dialysis bag. you can recover most of the protein from the gel.